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  • Open access
  • 12 Reads
Positive Pharmacological Modulation of Hsp70 in Recovery of Brain Energy Metabolism in Various Models of Cerebral Ischemia

In studies on Wistar white rats of different ages with various experimental models of cerebral ischemia (intracerebral hemorrhage, carotid artery occlusion, prenatal hypoxia), it was found that pharmacotherapy of emerging mitochondrial disorders with modulators of 70 kilodalton heat shock proteins (HSP70) expression - Cerebrocurin (composition: neuropeptides, S-100 proteins, reelin, factor nerve growth (NGF) ), Glutoredoxin, Tamoxifen, (S)-2,6-diaminohexanoic acid, 3-methyl-1,2,4-triazolyl-5-thioacetate significantly (p<0.05) stimulates energy production brain. This is expressed in an increase in the content of a number of intermediates of energy metabolism (glucose, pyruvate, succinate, isocitrate, malate) in the brain tissue, as well as an increase in the activity of pentose phosphate shunt dehydrogenase. A significant (p<0.05) decrease in the level of lactic acid in the brain indicates a decrease in the proportion of anaerobic glycolysis. The most pronounced energotropic effect was shown by cerebrocurin (150 µl/kg) and (S)-2,6-diaminohexanoic acid 3-methyl-1,2,4-triazolyl-5-thioacetate (50 mg/kg). On the 21st day of treatment of rats with cerebral ischemia, there is a gradual disappearance of severity between animals with simulated pathology and healthy rats, which is associated with actively recovering mitochondrial energy production and cerebral load in general. These changes in energy metabolism correlate with the normalization of oxidant-antioxidant balance and the concentration of HSP70 and HSF-1a in the cytosol and mitochondria of the cerebral ischemia zone and are manifested in a significant (p<0.05) decrease in ultrastructural disorders of mitochondria. The energy-tropic and mitoprotective effects of the studied drugs are associated with their ability to protect mitochondrial proteins from oxidative damage by increasing the concentration of HSP70, prolong the lifespan of HSF-1a, and activate alternative energy generation pathways.

  • Open access
  • 29 Reads
Self-Association Evidence for Antimicrobial Peptides via Long-Scale MD Simulations: a Case Study

Antimicrobial peptides (AMPs) are a diverse class of short proteins, that are a key element of the nonspecific innate immunity in most organisms, displaying a wide range of antimicrobial, antifungal, antiviral, and even anticancer effects. Lately, AMPs have attracted great research interest in the context of strategies to combat multi-drug resistant bacterial infections. AMPs come in nature in the form of multicomponent secretory fluids that exhibit certain biological activity. Although these small proteins are usually cationic and amphiphilic, their antimicrobial action is not completely understood, neither is their behavior in bodily liquids prior to attacking the target membrane. We studied various linear AMPs behavior in mono- and multicomponent solutions, prior to their engagement with the pathogenic membrane, by means of long-scale molecular dynamics simulations. We observed that the peptide monomers self-associate into clusters, which consist of a non-polar hydrophobic core and exposed to the solvent charged and polar residues. We consider the so-formed structures as the perfect transport system – locking the hydrophobic uncharged residues in the core of the cluster prevents the interaction with the uncharged eukaryotic membranes, and positioning the charged residues on the cluster surface enables electrostatic interaction with the bacterial surface. The very formation of the clusters but also the peptide folding, promoted by the amphiphilic structure in the aggregates, allow for an increase of the local concentration of AMPs to be delivered to the target membrane in a functionally active conformation.

Acknowledgments: This work was supported in part by the Bulgarian National Science Fund under Grant KP-06 OPR-03-10/2018. Computational resources were provided by BioSim HPC cluster at the Faculty of Physics, Sofia University “St. Kliment Ohridski” and by CI TASK (Centre of Informatics – Tricity Academic Supercomputer & networK), Gdansk (Poland).

  • Open access
  • 8 Reads
Levels of selected biochemical parameters in homogenates of isolated pig kidneys flushed with preservative solution supplemented with selenium and prolactin

Abstract: Selenium in the human body exhibits strong antioxidant and immunomodulatory properties. This bio-element is found in the active centers of antioxidant enzymes, which participate in the elimination of damage caused by free oxygen radicals. It enters into the composition of some proteins that build cell membranes, performing stabilizing functions. Its involvement in catalyzing redox reactions helps reduce oxidative stress and potentially minimize ischemia-reperfusion damage in the kidney during ischemia. The aim of this study was to determine whether supplementation of preservative fluid with selenium in the presence of antioxidant prolactin affects the levels of selected biochemical indices in homogenates of isolated porcine kidneys. The work is part of a series of our team studies to develop the optimal fluid composition for organ perfusion and preservation. Biolasol preservation fluid was modified by adding Se4+ (1 µg/L) and prolactin (0.1 µg/L). The study was conducted on 30 isolated kidneys of Polish Large White pigs. The kidneys were randomly divided into 3 groups (n=10 in each) and washed with preservative fluids: Biolasol - control kidneys (C), Biolasol+Se (A1), Biolasol+Se+PRL (A2). After 48h of preservation and perfusion, kidney sections were excised. Selected biochemical markers were determined in the tissue homogenates: protein and creatinine concentration. The study was performed in accordance with the recommendations of the II Local Ethics Commission for Animal Experiments in Cracow, Poland (number 1046/2013) and in accordance with the European Union Directive (EU guideline 93/119/EC). Supplementation of Biolasol solution with selenium and prolactin caused a statistically significant reduction in protein and creatinine levels compared to the control group in homogenates of isolated pig kidneys. Protein concentrations were: 2.5±0.1 mg/g (group C) vs 0.9±0.2 mg/g (group A2) (P<0.05); creatinine concentrations were: 2.5±0.1 mg/g (group C) vs 1.9±0.2 mg/g (group A2) (P<0.05). In contrast, the use of selenium alone (without PRL shielding) resulted in a statistical increase in marker concentrations. Protein concentrations were 20% higher compared to Biolasol (P<0.05); creatinine concentrations were 16% higher compared to Biolasol (P<0.05). Presumably, there was an accumulation of selenium in the organ, exacerbating the resulting damage. Selenium and prolactin added to Biolasol fluid show protective effects on nephrons. Selenium (IV) as a component of Biolasol solution adversely affects renal protection during ischemia.

  • Open access
  • 11 Reads
Co-expression of VEGF and FGF2 mediated by multigenic plasmid constructs promotes blood flow restoration in a rat model of hind limb ischemia.

Peripheral arterial disease (PAD) is characterized by stenosis and occlusion of the arteries leading to the poor blood supply to the limb. Patients with PAD suffer pain at rest, intermittent claudication, skin ulcers, and gangrene. The end-stage of the disease could lead to limb amputation despite optimal medical and surgical management. The delivery of angiogenic factors to restore tissue perfusion is an attractive strategy, both as a primary and adjunctive treatment for PAD.
We synthesized multigenic plasmid constructs expressing combinations of VEGF, FGF2, and DsRed genes:pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, pVax1-DsRed. In the constructed vectors, gene sequences are linked through the furin-containing 2A-peptide sequence of picornaviruses. Plasmid vector pVax1, approved by the FDA for use in clinical trials.
Previously, we confirmed the functionality of the developed non-viral constructs and the synthesis of VEGF, FGF2, and DsRed proteins by transfected cells. At this stage, we injected plasmid constructs into rat muscles after hind limb ischemia. Quantitative analysis of serum cytokines and chemokines of experimental and control groups on 3, 14, and 21 days after plasmids injection showed no significant differences in the secretion of the 18 cytokines studied. We observed a gradual increase in volumetric blood flow in the experimental groups, despite decreased expression of VEGF and FGF2 on 14 and 21 days. On day 21, the maximum increase in volumetric blood flow was in the pVax1-VEGF-FGF2-DsRed group. In turn, the maximum number of capillaries at 21 days was in the pVax1-VEGF-DsRed group. Capillary density was increased in pVax1-VEGF-FGF2-DsRed, and pVax1-FGF2-DsRed groups compared to control groups. We also observed low expression levels of caspase-3 and caspase-9 in the muscles of the experimental groups.
Thus, co-expression of VEGF and FGF2 stimulates angiogenic and regenerative processes in a rat model of hind limb ischemia. In addition, the results of this study are consistent with our previous work and confirm the effectiveness of using systems based on 2A-peptide sequences for transgene co-expression. The study of the serum cytokine profile showed the absence of adverse immune effects, indicating the safety of the non-viral constructs used. These results suggest the possibility of using these non-viral constructs to enhance therapeutic angiogenesis in the treatment of ischemic diseases. This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

  • Open access
  • 24 Reads
The first opsins prediction in Achatina fulica genome

The variety of spectral properties of visual pigments is provided mainly by the amino acid sequences of their protein part, the so-called visual opsins. However, such data on these proteins of gastropod molluscs are very scarce, and for terrestrial species are absent at all. Meanwhile, the first sequenced genome of the terrestrial gastropod, Achatina fulica, was published ( So, the aim of this study is the prediction of the amino acid sequences of opsins in the A. fulica genome.

To this purpose, all known mollusc opsin sequences from the NCBI Protein database were collected. To analyse them, we performed a multiple sequence alignment in Unipro UGENE by Clustal Omega and refined it by MUSCLE, and then constructed the HMM profile, which was used to predict opsins in the A. fulica genome. Prediction of opsin sequences was performed using HMMER. To confirm the obtained sequences belong to opsins, we searched for the conserved domain, 7tmA_Opsin_Gq_invertebrates.

As a result, we determined five rhodopsin G-coupled protein sequences in A. fulica genome among them are Afu011750, Afu004575, Afu005002, Afu003765, Afu022267.

This is the first attempt to annotate the Achatina fulica genome, which was started by searching for opsins. Our study opens up new opportunities for further more detailed evolutionary and genetic studies of molluscs, including the study of visual mechanisms. But it should be noted that opsins form a diverse multifunctional group of proteins, of which only some are part of visual pigments. Therefore, work towards the identification of visual opsins in Achatina fulica should be continued.

  • Open access
  • 5 Reads
DNA obtained by ab initio synthesis forms hyperbranched net-like structure

Ab initio DNA synthesis is unusual synthesis of dsDNA from tens bp to kbp long by thermophilic DNA polymerases from free dNTPs in the complete absence of added DNAs. As commonly believed, the reaction product is a linear double-stranded DNA in the B form. However, an extremely low efficiency of cloning and the failure to hydrolyze high-molecular-weight DNA, as well as the presence short repeats, palindromes, and AT-rich repeats in the sequence assumes more complex spatial structure of this DNA. The AFM coupled with nuclease analysis revealed that high-molecular-weight dsDNA products branched and formed net-like structures. The DNA contained single-stranded and triple-stranded segments. These net-like structures may be assumed to be three-dimensional (3D). The present work was the first detailed investigation of the ab initio synthesis products. The results may be useful to develop techniques requiring synthesis of large amounts of DNA with complex spatial structure.

  • Open access
  • 6 Reads
Validation and amplification of random DNA libraries with modified nucleobases for Click-SELEX

Aptamers are nucleic acid ligands which display specific binding to a desired target. Oligonucleotides are limited in their ability to form hydrophobic interactions. The affinity of aptamers can be improved with the introduction of hydrophobic modifiers into the structure of nitrogenous bases. Click-SELEX is a powerful tool for the development of the aptamers containing modified nucleobases. This technology assumes adding of azide-bearing modifier of choice to alkyne-modified random DNA library using click-chemistry. The synthesis and amplification of modified libraries is a limitation for performing click-SELEX. This research is aimed to validate the modified DNA libraries using NGS and to study specific aspects of their amplification. Commercially available library with alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU) instead of thymidine was obtained in four variants with different distribution of nucleobases. Alkyne-containing (before clicking of a hydrophobic moiety) and azide-modified (after clicking) libraries were amplified with four different DNA polymerases. All the enzymes screened were able to amplify non-natural DNA template, the best amplification efficiency was shown for Taq DNA polymerase. NGS of alkyne-containing libraries confirmed the correct length, high diversity of the libraries and the uniformity of nucleotide distribution per position. No significant difference between the sequenced samples amplified with two different DNA polymerases was recognized. In all four libraries the content of EdU was lower than it was assumed during the chemical synthesis; 14, 13.0, 16.4 and 18.5% EdU was detected in the random core instead of 20, 20.4, 23.1 and 25% of theoretical EdU content. To reach the equimolar distribution of nucleobases, a higher proportion (more than 25%) of EdU should be used during the chemical synthesis of the library. We believe these results provide an experimental basis for the expansion of click-SELEX technology to the routine aptamer research.

The study was supported by the Russian Science Foundation grant No. 21-79-10175,

  • Open access
  • 79 Reads
Graphene: A new material for wound healing

One of the hot topics in medical research involves using new materials for repairing human tissues. Graphene and its derivatives (which would be abbreviated as graphene in the following text for simplicity) happens to be one such novel material and has excellent properties due to ability to help regenerate tissues. This could be potentially useful for tissue engineering, and skin/muscle/nerve/bone/cartilage repair. Wound healing in eczema, bed sores or burning accidents have a high risk of bacterial infection, and some graphene materials have shown to provide good therapeutic efficacy in the improvement of wound healing with new dressing materials. The mechanism of wound healing might be due to its anti-bacterial properties, immunomodulatory effect, anti-inflammatory effect as well as angiogenic properties. The reason behind this unique properties could be its lack of cytotoxicity, its compatibility with biological material in terms of adhesiveness, and its lack of inhibition of healthy cell migration. Graphene could also have some antibacterial properties which might be due to its dehydrating properties or the ability of some functional groups to generate free radicals that kill pathogenic bacteria. The biocompatibility could be demonstrated by examination of the adhesion of fibroblasts cell lines and the morphology of their filopodia (or the feet). While many model systems concentrate on skin repair and wound healing, the use of graphene is not limited to skin cells: A human stem cell model has also been used to mimic cell regeneration from acute myocardial infarction (MI) using graphene in the form of hydrogels. This model has established the enhanced cell survival rate, increased expression of pro-inflammatory factors, factors that aid in the formation of new blood vessels, and early cardiogenic biomarkers, using graphene quantum dots as a soft injectable hydrogel for heart regenerative function after MI. The study of graphene and graphene-based materials on inflammatory biomarkers and acute phase proteins will be the subject of investigation in this review.

  • Open access
  • 18 Reads
Phytochemical Screening and Antioxidant activity of Trichosanthes cucumerina , Momordica charantia var muricata and Luffa acutangula.
, , , , , ,

Abstract: Background- TC, LA, MCM, plants (Family: Cucurbitaceae) are widely used in traditional medicine and an important source of vegetables in the world. Objective- This study aimed to evaluate the phytochemical constituents of the 95% ethanolic extract obtained from the TC, LA, MCM whole plants as well as to study the antioxidant activity by DPPH assay. Materials and Methods- Mechanically plant powdered extracted in the soxhlet apparatus & phytochemical screening, DPPH assay performed by following the standard protocols. Results- The phytochemical screening revealed the presence of Phenols & Steroids in all TC, LA, MCM whole plants, Alkaloids in TC, MC, Saponins in MC, LA and Glycosides only in TC plant. The percentage of the inhibition and IC50 were measured, results showed that the DPPH free radicals were scavenged by all extract in a concentration dependent manner. Conclusion- These dietary cucurbits showed appreciable antioxidant activity and are good sources of natural antioxidants. Future pharmaceutical uses can be deducted from these findings.

Abbreviations: TC: Trichosanthes cucumerina, LA: Luffa acutangula, MCM: Momordica charantia- muricata DPPH: 2 , 2-Diphenyl-1-picrylhydrazyl

  • Open access
  • 15 Reads
In silico determination of changes in transcription factor binding sites for the preeclampsia risk haplotype in the regulatory region of the FLT1 gene

Preeclampsia (PE) is one of the most common complications of pregnancy that occurs in 3-8% of pregnant women, being one of the top five causes of maternal morbidity and mortality. We found that PE-associated polymorphisms near the FLT1 gene are located in the same regulatory region and together can be a genetic pattern that affects the development of pathology, forming a PE risk haplotype.

We used in silico study design, using for database analysis. Under examination PubMed and GWAS-catalog we chose only SNPs located in FLT1 gene and it's regulatory area with association p-value threshold of 5 × 108. All SNPs in regulatory region were selected with MAF greater than 1 % for exclusions from the analysis of rare genetic variants. Haplotype of risk calculated with help LDhap Tool for EUR populations, including SEU,TSI, FIN, GBR, IBS based on the alleles frequency of the analyzed polymorphisms. Possible haplotype risk were determined according to PE-associated alleles of polymorphisms (rs4769612 and rs7318880), as well as comparison of the prevalence of haplotypes with the prevalence of PE in the European population. Under TFBs changes analytics in HumanTFDB we use p-value cutoff 1 × 10−4 when analyzing TFBs for all possible haplotypes. Determination of TF expression in placenta were performed with Proteomic DB database search.

We used GWAS-catalog to identify polymorphisms rs4769612 and rs7318880 in FLT1 associated with preeclampsia in the maternal and child genomes. According to UCSC genome browser (oRegAnno) 5 regulatory elements overlap: OREG1191996, OREG1658246, OREG1688336, OREG1537828 and EH38E1663332. In the regulatory region, we selected SNPs with a MAF of 1% and identified possible haplotypes in European populations using LDhap Tool. We identified 4 possible haplotypes, with frequency theoretical risk haplotype 8.25% (rs7320190-C, rs7318880-T, rs12867370-A, rs4769612-C, rs4769613-C, rs74623647-G, rs7321138-C, rs76592233-C).

Using HumanTFDB, we identified changes from 83 transcription factor binding sites (TFBs) to minus DNA strands in the analysis of all 4 possible haplotypes. Only 40 transcription factors (TFs) are expressed in the placenta, according to Proteomic DB. When analyzing the changes in TPFS, which are characteristic only for the risk haplotype with a prevalence in the European population of 0.0825, we found that 5 TPTFs change. At the same time, the number of TFBSs for ELF1, SPIB increases. The number of TFBS POLR2A, KLF15 decreases (not expressed in the placenta). The most significant is the emergence of a new TFBS KAT5, for the promoter of which only a DNase signature is observed until day 118 of pregnancy in the placenta, and after day 118 it acquires a promoter signature. Theoretically, the appearance of a new TFBS can increase the expression of FLT1, causing an imbalance of angiogenic - antiangiogenic factors, characteristic of PE.